ABSTRACT Although in general, the larger the primary tumor the greater the likelihood that it will metastasize or already has metastasized, this is not always the case. Many small breast cancers develop metastasis and have a discouraging outcome. Characterizing genes that drive these tumors' rapid progression may identify novel biomarkers to help clinicians guide current treatments, and may offer novel therapeutic targets. Our work in the prior funding period focused on the role of the matricellular protein CCN6 in breast tumorigenesis, which was unknown. We have convincingly demonstrated that while CCN6 protein is expressed in breast epithelial cells, it is reduced or lost in 60% of invasive breast carcinomas, where it is associated with poor outcome. We have clearly shown that decreases invasion and metastasis in breast cancer in vivo and in vitro. Our preliminary data and the current proposal are consistent with the long-term objectives of this laboratory: to understand basic mechanisms associated with breast cancer progression and translate them into clinical utility. We have shown that downregulation of CCN6 in nontumorigenic breast cells disrupts normal acinar organization and triggers an epithelial to mesenchymal transition (EMT) and invasion, which requires BMP4-mediated activation of TAK1 and p38 pathway, known to promote metastasis. Our laboratory has provided the first mechanistic link between CCN6 and BMP4 signaling in humans. CCN6 binds to BMP4 protein in breast cancer cells and reduces invasion. However, the mechanistic details and in vivo consequences of this interaction to breast cancer metastasis need further investigation. The overall goal of the current proposal is to determine the potential mechanisms whereby CCN6 reduces breast cancer metastasis and to translate biologic findings into the clinical field. The central hypothesis is that CCN6 suppresses breast cancer progression by binding to BMP4 and antagonizing the effect of BMP4-mediated activation of TAK1 and p38 kinases on EMT, invasion, migration, and metastasis. We propose the following three hypothesis-driven specific aims. AIM 1: To investigate the in vivo consequences of CCN6 conditional knockout in the mammary gland and to define the critical CCN6 protein motifs involved in breast cancer progression. AIM 2: To determine the mechanism of CCN6-mediated metastasis suppressor function in vivo and its relationship to BMP4/TAK1/p38 signaling. AIM 3: To ascertain the translational impact of CCN6 and the BMP4 pathway in human breast tissue samples. We will investigate the effect of CCN6 on the EMT, invasion and metastasis and on BMP4/TAK/p38 signaling in vivo using unique human tissue resources. Regulating the expression of CCN6 may represent an important drug target for prevention and/or reduction of breast cancer metastasis. With more than 1.2 million women diagnosed this year worldwide, these studies have the potential of providing better diagnosis, more accurate prognostication, and pave the way for future development of small molecules based on CCN6.